Obesity correlates with an increased risk of a range of diseases including some types of cancer, heart disease and type two diabetes. Recently it has been reported that the presence of specific alleles in the FTO gene, located on human chromosome 16, correlates with obesity and type two diabetes. Specifically, sufferers of type two diabetes were found to have an increased likelihood of a particular FTO variant (rs9939609 A allele) which correlates with an increased body weight (Frayling et al. Science (2007) 316: 889-894). Subsequent analyses involving more than 39,000 individuals revealed that the FTO allele was associated with body weight. Individuals carrying one copy of the FTO variant associated with type two diabetes had a 30% increased risk of being obese compared to an individual with no copies of that version and were on average more than 1.2 kg heavier than individuals with no copies of the disease-associated variant. Individuals with two copies of the variant (about 16% of those analyzed) had a 70% increased risk of being obese and on average weighed 3 kg more than individuals with no copies of the disease-associated variant. This study is important, as no previous work has identified a risk allele for obesity that is so prevalent. Further studies show similar results (Dina et al. Nat Genet (2007) 39: 724-726; Scott et al. Science (2007) 316: 1341-1345).
The mouse ortholog of FTO, Fatso (Fto), is a gene that is deleted in the fused toes mouse mutant (Peters et al. Mann Genome (2002) 13: 186-188; van der Hoeven et al. Development (1994) 120: 1601-2607, Grotewold & Ruther, Dev Biol (2002) 251: 129-141; Anselme et al. Dev Biol (2007) 304: 208-220). For clarity, subsequent reference to FTO includes human FTO, non-human homologues and/or any of their clinically observed variations.
Although FTO sequence variation is linked to obesity, the function of its protein product at the biochemical, cellular and physiological levels has not been reported.
Knowledge about FTO structure and its biochemical, cellular and physiological roles is needed to enable the correlation between FTO and obesity to be exploited for the treatment of diseases associated with weight gain, such as diabetes, cardiovascular disease, cancer, osteoporosis and hypertenstion.
The 2-oxoglutarate (2-OG) and ferrous iron dependent oxygenases are a superfamily of enzymes that catalyse a wide range of reactions including hydroxylations, desaturations and oxidative ring closures (Hausinger (2004), Crit. Rev. Biochem. Mol. Biol. 39, 21-68; Ryle & Hausinger (2002) Curr. Opin. Chem. Biol. 6, 193-201; and Schofield et al. (1999) Journal of Inorganic Biochemistry 74, 49-49). Substrate oxidation is coupled to conversion of 2-OG to succinate and carbon dioxide. At least in some cases, binding of oxygen is followed by the oxidative decarboxylation of 2-OG to give succinate, CO2 and a ferryl species [Fe(IV)=O] at the iron centre. This highly reactive intermediate can then oxidize an unactivated C—H bond in the prime substrate, e.g. the oxidation of prolyl or asparaginyl residues in human proteins, or effect other oxidative reactions such as oxidation of methyl groups on N-methylated versions of proteins or nucleic acids. Evidence for intermediates comes from substrate-analogue studies, model compounds and spectroscopic analyses.
The sequential binding of co-substrate and prime substrate, which is necessary to trigger oxygen binding, is probably important in limiting the generation of reactive oxidizing species in the absence of prime substrate. The generation of such species in a prime-substrate-uncoupled manner can inactivate 2-OG and the related oxygenases through self-oxidation, which sometimes leads to fragmentation. Typically, the uncoupled turnover of 2-OG occurs at approximately 5% of the rate of its coupled turnover in the presence of saturating concentrations of prime substrate, although it can also occur at a lower or higher rate.
Several 2-OG-dependent oxygenases, including procollagen prolyl hydroxylase, the hypoxia inducible factor prolyl hydroxylases, and anthocyanidin synthase, have a requirement for ascorbate for full catalytic activity. Although ascorbate might stimulate activity by reducing Fe3+, or other high valent forms of iron, to Fe2+ (either free in solution or at the active site), the stimulation of oxygenase activity by ascorbate might occur by other mechanisms, for instance, by promoting completion of uncoupled cycles. For uncoupled reaction cycles that are catalysed by procollagen prolyl hydroxylase in the absence of prime substrate, the oxidation of 2-OG to succinate has been shown to be stoichiometrically coupled to ascorbate.
Furthermore, the activity of ascorbate has been shown to stimulate the activity of 2-OG oxygenases in cells (e.g. in work on the hypoxia inducible factor (HIF) prolyl hydroxylase) and lack of ascorbate in the human diet leads to the disease scurvy due to impaired activity of the procollagen prolyl hydroxylase.
Studies with several enzymes have shown that certain substrate analogues and mutants can also stimulate uncoupled 2-OG turnover. It is also known in the literature that reducing agents other than ascorbate itself can act as reducing agents in the uncoupled turnover reaction, including derivatives of ascorbate (Zhang et al. (1995) Biochem. J. 307 (Pt 1), 77-85 and Myllyla et al. (1978) Biochem. Biophys. Res. Commun. 83, 441-8).
A number of 2-OG oxygenases are of current therapeutic interest including the transcription factor hydroxylases, e.g. the hypoxia inducible factor prolyl and asparaginyl hydroxylases, methylated nucleic acid demethylases, methylated lysyl demethylases, procollagen prolyl and lysyl hydroxylases, phytanoyl CoA hydroxylase, Mina53, NO66 and arginyl hydroxylases such as phosphatidyl serine receptor (Jmjd6). The methyl lysyl demethylases may use methylated histones as preferred substrates and may use any of all the tri, di, or mono-methylated lysine residues as preferred substrates.
Methylation of nucleic acid and nucleic acid associated acid proteins, including histones, is a known mechanism for epigenetic inheritance. Methylation of nucleic acids can also affect gene activity and expression.